Automated protein analyzer

ABSTRACT

A direct rapid automated protein analyzer is disclosed. The protein analyzer includes means for reducing protein samples to small particles, a reaction vessel in material transfer communication with the homogenizer, a reservoir for binding dye composition in fluid communication with the reaction vessel, a metering pump in fluid communication with the reservoir and the reaction vessel for distributing discrete predetermined amounts of a binding dye composition to the reaction vessel, a filter in fluid communication with the reaction vessel for separating solids from filtrate after a dye binding reaction has taken place in the reaction vessel, and a calorimeter in fluid communication with the filter and the reaction vessel for measuring the absorbance of the filtrate from the reaction vessel and the filter. The rapid analyzer can be used in conjunction with a kit that includes a sample cup for mixing a protein sample with a dye-binding solution and a filter holder for being positioned in the sample cup. The filter holder includes a filter media and a depending spout below the filter media that reaches bottom portions of the cup when the filter media is positioned above the cup. The kit can also include dye concentrate solution.

BACKGROUND

The present invention relates to the determination of proteins inmaterials and particularly the protein content in various food samples.

Proteins are long chain molecules formed from the 20 basic amino acidsand are the building blocks of all living systems. Proteins alsorepresent, along with carbohydrates, fats and oils, a required foodsource for almost all living things.

Because proteins are a required food source, they are widely availablein commercially available food products. Human beings tend to takeprotein in the form of meat, poultry, eggs seafood, dairy products, andnuts. Proteins are also a necessary part of many animal diets, includingfarm animals raised commercially. Protein sources for such animal feedscan also include meat, poultry, eggs, fish, and grains such as corn andoats.

Because so much human and animal food moves through a fairlysophisticated growing and distribution system, the knowledge of theamount of protein in food products is a valuable or even necessary forquality control, manufacture, storage, distribution, and use. As aresult, the need to measure the protein content of various food productsfor both human and animal consumption has long existed.

One original (although indirect) test for protein content is theKjeldahl test for nitrogen. In this test a protein sample is mixed withdigestion ingredients (e.g., concentrated sulfuric acid, H₂SO₄) andoften in the presence of mercuric oxide catalyst, potassium sulfate, andhydrogen peroxide. The acid converts the nitrogen into ammonium sulfate.The resulting solution is then made alkaline, liberating ammonia. Theamount of ammonia can then be determined by titration with standard acidor any other relevant technique. A microwave instrument and techniquefor Kjeldahl analysis is set forth in commonly assigned U.S. Pat. No.4,882,286.

Although the Kjeldahl test offers the advantage of determining proteincontent, it does so based on total nitrogen rather than protein per se.Thus, any given test results can include nitrogen from sources otherthan proteins, peptides, or amino acids. The Kjeldahl test also requiresheating the sulfuric acid to temperatures that can reach 300° C. and inthe presence of a metal catalyst. The Kjeldahl test is relativelycomplex, can take as long as 4 or 5 hours and can be susceptible tofalse nitrogen results. In the latter circumstance, confirmationrequires at least a second test.

The Dumas technique presents an alternative analysis for total nitrogenand total carbon analysis. This is a combustion technique based upon thegeneration of gas phase products by extremely rapid combustion of thesample material. In an exemplary technique, a sample is carried in a tincombustion capsule and dropped into a combustion chamber that includes acatalyst and that is maintained at a relatively high temperature (1200°C.). A pulse of pure oxygen is admitted with the sample and the thermalenergy from the resulting combustion of oxygen and tin generates aninstantaneous temperature of as high as 1700° C. The heat produces totalcombustion of the relevant materials and the resulting gas phaseproducts are collected in a stream of inert gas such as helium.Alternatively, the sample can be oxidized in the presence of a hot metaloxide. Carbon in the sample is converted to carbon dioxide (CO₂). Thenitrogen combustion products include diatomic nitrogen (N₂) and thevarious oxides of nitrogen. These are directed through a reductioncolumn, typically using heated metallic copper, to reduce the nitrogenoxides to diatomic nitrogen. The nitrogen can be determined from thevolume of N₂ produced or by other comparative techniques such as thermalconductivity measurements.

The Dumas technique is limited to relatively small sample sizes (e.g.0.5 grams or less) and like the Kjeldahl technique it is indirectbecause it measures total nitrogen rather than protein per se. The smallsample size also makes the Dumas test less suitable for moreheterogeneous materials.

Indirect techniques such as infrared or near infrared spectroscopy canbe used but require relatively extensive calibration. Additionally, thepresence of water tends to obscure the infrared absorption across arelatively wide portion of the spectrum. Because plant and animalproteins are so often found in the presence of at least some water,these infrared techniques are often inefficient.

For these and other reasons, proteins are sometimes measured by adye-binding method, an original version of which was developed by DoyleUdy; e.g., “A Rapid Method for Estimating Total Protein in Milk,”Nature, Vol. 178, pp 314-315, Aug. 11, 1956.

In a simplified description, a protein sample, usually in liquidsuspension, is mixed at an appropriate pH with an aqueous solution of adye molecule that will bind to the proteins. The solution contains anexcess of the dye based upon the expected protein content of the sample.Proteins and these specified dyes react to form precipitated solids thatremove the dye molecules from the solution. The solution is thenfiltered from the precipitate. The loss of color in the filtrate asmeasured in a spectrometer or calorimeter is proportional to the amountof dye (and thus protein) that formed the precipitate. This can also beexpressed as the filtrate color being inversely proportional to theprotein concentration (i.e., the higher the protein concentration theless color in the filtrate). As a typical example, a solution containingacid orange 12 dye (crocein orange G) has a readily identified broadabsorption peak at about 482 nanometers (nm) and its absorbance followsBeer's Law.

As one advantage of this technique, the dye binds strongly with proteins(amino acids) rather than other nitrogen-containing compounds. Thus, itmeasures protein content more directly than do the nitrogen contenttechniques.

The technique does, however, require relatively complex measurement andhandling techniques, or at least a plurality of manipulative steps eachof which must be carried out properly in order to get an accurateresult. For example, the user must prepare samples carefully because thesmall portions tested often represent much larger selections(potentially tons) of non-uniform materials. The test is generallycarried out on suspensions which must be handled and stored and preparedappropriately. When solid materials are tested, they must typically beground or pulverized to obtain an appropriate sample. Semi-solidmaterials tend to vary in their uniformity with some being almosthomogeneous and others being quite non-homogeneous. When samples cannotbe used immediately, preserving them for longer periods of time requiressignificant care.

The reagents present additional challenges and must be carefully handledin preparation, storage, and use. The accuracy requirements of solutionpreparation are relatively stringent and the preparations must becarried out appropriately.

In conventional practice, mixing an insoluble protein sample directlywith a dye binding solution produces a heterogeneous mixture of theoriginal sample, the dye-protein precipitate, and the remaining dyesolution. This mixture is typically full of solids both from the dyebinding reaction and the original sample and is generally too unwieldyfor the necessary filtration and colorimetry steps. As a result,conventional dye-binding techniques tend to avoid directly mixing thedye solution and the protein sample (typically a food product).Instead—and in an additional step—a carefully weighed sample of proteinis first diluted in measured fashion to about 10 times its originalvolume typically with water, or water, methanol and citric acid(citation). This diluted mixture is then blended to form a morehomogenous diluted sample. The homogenized diluted sample is then mixedwith the dye binding solution to initiate the dye-binding reaction.

As result, the necessary dilution introduces an additional manipulativestep, an additional measurement step, and an additional calculation intothe overall process.

Protein testing usually involves obtaining and preparing severaldifferent sets of the acid orange 12 dye. For example, in the basic Udytechnique (Udy Corporation, Principles of Protein Measurement,http://www.udyone.com/udydocs/udysys2.shtml, accessed May 7, 2007) thefiltrate color is measured using a digital calorimeter. The calorimeteris set using a reagent dye solution and a working reference dyesolution. The standard reference dye solution is used to verify theproper concentration of the reagent dye solution and of the workingreference dye solution. The reagent dye solution and the standardreference dye solutions are available in prepared format or asconcentrates which can be diluted with distilled water and acetic acidbefore use. The user prepares a working reference dye solution from thereagent dye solution.

Stated more simply, the amount of protein in a given sample is measuredby comparing the “before and after” color of the dye solution. Becausethe “before” color of any given solution can vary slightly dependingupon its preparation, the calorimeter must be calibrated to match theindividual dye solution before every test or before a series of teststhat use that dye solution.

These relatively strict requirements produce good results, but the manysteps involved compound the normally expected experimental uncertaintyand each step also introduces the potential for outright error.

For example, typical dye binding protein sampling kits include ablender, a separate container and valves for the dye solution, aseparate filter for separating the protein-dye precipitate from thefiltrate, and a separate calorimeter. In the same manner, the “basicsteps” of protein determination of meat products include the initialdilution step, then homogenizing the diluted sample in the blender,removing the sample from the blender with a syringe, a pipette, or bypouring it into a bottle; adding and measuring the reagent dye solutionto the sample; shaking the sample; and filtering the reaction productinto the calorimeter to read the absorbance, or in some cases asoftware-generated protein content based upon the absorbance (UdyCorporation, Udy Protein Systems, www.udyone.com/prosysinfo.htm,accessed Aug. 7, 2007).

These testing steps must be preceded by similarly strict steps forpreparing standardized dye solutions for both reference (calibration)and testing purposes.

In the 1970's Foss (a/k/a Foss America, Foss Electric and Foss NorthAmerica) offered a dye-binding test for milk in the form of the“Pro-Milk II” system. More recently, however, Foss has developed andoffered automated devices that use either Kjeldahl techniques orinfrared spectroscopy to measure protein content in milk products; e.g.,Foss North America, Products direct, (online)http://www.foss.us/solutions/productsdirect.aspx (accessed July 2007).

Accordingly, a need exists for protein measurement techniques thatminimize or eliminate these disadvantages.

SUMMARY

In one aspect the invention is a direct rapid automated proteinanalyzer. In this aspect the invention includes a homogenizer forreducing protein samples to small particles, a reaction vessel inmaterial transfer communication with the homogenizer, a reservoir forbinding dye composition in fluid communication with the reaction vessel,a metering pump between the reaction vessel and the reservoir fordistributing discrete predetermined amounts of a binding dye compositionto the reaction vessel, a filter in fluid communication with thereaction vessel for separating solids from filtrate after a dye bindingreaction has taken place in the reaction vessel, and a calorimeter influid communication with the filter and the reaction vessel formeasuring the absorbance of the filtrate from the reaction vessel andthe filter.

In another aspect the invention is a dye binding method for proteinanalysis. The method includes the steps of preparing an initialreference dye solution of unknown concentration from an initialreference dye concentrate, creating an electronic signal based upon theabsorbance of the initial reference dye solution, thereafter creating anelectronic signal based upon the absorbance of a dye filtrate solutionprepared from the initial reference dye solution and an initial proteinsample, sending the absorbance signals from the reference dye solutionand the dye filtrate solution to a processor that compares therespective absorbances and calculates the protein content of the proteinsample based upon the difference between the absorbances, creating anelectronic signal based upon the absorbance of a successive dye filtratesolution prepared from the reference dye solution and a successiveprotein sample, and sending the absorbance signal from the successivesample dye filtrate solution to the processor to calculate the proteincontent of the successive sample based upon the difference between theabsorbance of the initial reference dye solution and the absorbance ofthe successive dye filtrate solution.

In yet another aspect, the invention is an automated protein analyzerthat includes a reservoir for protein binding dye compositions, aprotein-dye reaction vessel in fluid communication with the dyereservoir, a calorimeter in fluid communication with the reservoir, apump in fluid communication with the reservoir and at least one of thecalorimeter and the reaction vessel for transferring dye compositionsfrom the reservoir to at least one of the calorimeter and the reactionvessel, a processor in signal communication with the calorimeter forreceiving the absorbance output from the calorimeter, and memory insignal communication with the processor for storing output from thecalorimeter that includes absorbance. The processor can compare thebaseline absorbance of a reference binding dye composition to thespecific absorbance of a binding dye composition following reaction witha protein to thereby calculate and determine the amount of protein in aprotein sample based upon the difference between the absorbance of thereference dye and the absorbance of the reference dye after it hasreacted with a protein sample.

In yet another aspect the invention is a method of calibrating acalorimeter for protein analysis and for analyzing a plurality ofproteins samples. In this aspect, the method includes the steps ofpreparing an initial reference dye solution of unknown concentrationfrom an initial reference dye concentrate and an approximate amount offluid, forwarding the initial reference dye solution to a calorimeterand measuring the absorbency of the reference dye solution, thereafterforwarding an initial dye filtrate solution prepared from the reactionof the initial reference dye solution and a protein sample to thecalorimeter and measuring the absorbency of the initial dye filtratesolution, sending the absorbance results from the initial dye filtratesolution and the initial reference dye solution to a processor thatcompares the respective absorbance and calculates the protein content ofthe sample based upon the difference between the absorbances, forwardinga successive dye filtrate solution to the calorimeter and measuring theabsorbency of the successive dye filtrate solution, and sending theabsorbance results from the successive dye filtrate solution to theprocessor to calculate the protein content of the successive samplebased upon the difference between the absorbance of the initialreference dye solution and the absorbance of the successive dye filtratesolution.

In another aspect, the invention is an improvement in the dye bindingmethod of protein analysis that includes the steps of mixing andhomogenizing a non-homogeneous, insoluble protein sample directly with adye-binding solution, drawing and filtering the remaining unreacted dyesolution directly from the homogenized mixture of protein sample anddye-binding solution, and measuring the absorbance of the filtrate.

In another aspect, the invention is a protein analysis kit that includesa sample cup for mixing a protein sample with a dye-binding solution anda filter holder for being positioned in the sample cup. The filterholder includes a filter media and a depending spout below the filtermedia that reaches bottom portions of the cup when the filter media ispositioned above the cup.

In yet another aspect, the invention is a protein analysis method thatincludes the steps of mixing a binding dye composition with a proteinsample, attaching a filter to a calorimeter, pumping unreacted dyecomposition from the mixture, through the filter and to the calorimeterwhile the filter is attached to the calorimeter, and measuring theabsorbance of the filtered dye composition in the colorimeter.

The foregoing and other objects and advantages of the invention and themanner in which the same are accomplished will become clearer based onthe followed detailed description taken in conjunction with theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an instrument according to the presentinvention.

FIGS. 2, 3 and 4 are respective cross-sectional, perspective, andexploded cross-sectional views of the filter holder and filter media.

FIG. 5 is a cross-sectional view of a sample cup according to thepresent invention.

FIG. 6 is a perspective view of a sample cup according to the presentinvention.

FIG. 7 is a perspective view of the turntable, homogenizer, and opticalsampling components of the invention.

FIGS. 8-13 illustrate the same components as FIG. 7, but additionallyillustrating the respective positions and movement of the sample cup,the filter, and the optical system during a protein analysismeasurement.

FIG. 14 is an exemplary normalized plot of absorbance versus proteincontent for measurements according to the present invention.

FIG. 15 is a perspective view of the analyzer according to the inventionin the context of its housing.

FIG. 16 is a perspective view of a protein analysis kit according to thepresent invention.

DETAILED DESCRIPTION

The present invention is an instrument and associated method for directand rapid dye binding protein analysis. The terminology used in thisspecification and the claims is generally clear in context. As a helpfulsummary, however, some common terms are used in the following manner.

The term “reference dye concentrate” refers to a pre-prepared (typicallycommercially prepared) concentrated solution of a reference dye thatwill bind with a protein to form a protein-dye precipitate.

In use, a reference dye concentrate is mixed with an appropriate amountof water (and potentially other items as described later herein) to forma reference dye solution. In the protein analysis testing, the referencedye solution is mixed with a protein sample.

The term “initial reference dye concentrate” refers to a reference dyeconcentrate that is used to prepare an initial reference dye solution.In turn, the initial reference dye solution is used in the first of aseries of protein analysis tests. The term “successive reference dyeconcentrate” refers to a second or further dye concentrate that is usedto prepare a second or further reference dye solution. The successivereference dye solution is used in additional protein analysis tests. Theinitial dye reference concentrate and the successive reference dyeconcentrate can be the same dye.

The term “initial protein sample” refers to the earliest in a givenseries of protein samples that are tested according to the method. Inthe same manner, the term “successive protein sample” refers to a secondor further member of a series of protein samples that are testedaccording to the method.

The term “dye filtrate solution” refers to the solution that remainsafter a protein sample has reacted with a reference dye solution. Inturn, the “initial dye filtrate solution” represents the filtrateobtained after a first of several (or many) reactions between a proteinsample and a reference dye solution. In the same manner, the term“successive dye filtrate solution” refers to the filtrate obtained afterthe second or further of several (or many) reactions between a proteinsample and a reference dye solution.

FIG. 1 is a schematic diagram of the elements of the instrumentaccording to the invention. It will be understood that FIG. 1illustrates the main functional elements of the instrument and thatalternative arrangements of these elements can still fall within thescope of the invention and of the claims. FIG. 1 illustrates a sampleholder or cup 10 which, as will be discussed with respect to the methodaspects of the invention can be weighed (tared) prior to adding aprotein sample. FIG. 1 illustrates a balance 19 for this purpose. Theinstrument transfers the sample from the cup 10 to a homogenizer broadlydesignated at 11. The homogenizer reduces the protein sample toparticles that are as small as possible to thereby provide for acomplete reaction with the binding dye. Accordingly, FIG. 1schematically illustrates the homogenizer 11 as including a blender 12or a ball mill 13. These are exemplary, however, and the homogenizer isnot limited to these specific types of equipment.

A reaction vessel 14 is in material transfer communication with thehomogenizer 11 as indicated by the line 15. As illustrated and describedwith respect to FIGS. 7-13, the functions of the cup 10 and the reactionvessel 14 can also be carried out using a single vessel by using ahomogenizer 11 that can be inserted into the cup 10 and then removed oncommand. A reservoir 16 for the binding dye composition is in fluidcommunication with the reaction vessel 14 through the line 17. Ametering pump 20 is positioned between the reaction vessel 14 and thereservoir 16 for distributing discrete predetermined (premeasured)amounts of the binding dye composition to the reaction vessel 14. Inorder to help drive the protein-dye reaction to completion, theinstrument can include an appropriate agitator, shown as the stirrer 18in FIG. 1.

A filter 21 is in fluid communication with the reaction vessel 14 forseparating solids from filtrate after a dye binding reaction has takenplace in the reaction vessel 14. A colorimeter broadly designated at 22is in fluid communication with the filter 21 and the reaction vessel 14for measuring the absorbance of the filtrate from the reaction vessel 14that passes through the filter 21.

FIG. 1 also illustrates a valve 23 between the reservoir 16 and thereaction vessel 14 as well as a fluid line 24 between the valve 23 andthe colorimeter 22. The combination of the valve 23 and the fluid line24 permit binding dye solution from the reservoir to be directed to thereaction vessel 14 or directly to the calorimeter 22. This provides theinstrument with the capacity to automatically make the referencemeasurements described in more detail with respect to the method aspectsof the invention.

The illustrated embodiment also includes an optics pump 25 between thereaction vessel 14 and the colorimeter 22 for transferring filtrate fromthe reaction vessel to the calorimeter 22.

The nature and operation of a colorimeter is generally well understoodin this art and will not be described in detail other than toschematically note as in FIG. 1 that the colorimeter 22 includes a lightsource shown as the diode 26, a photodetector shown as another diode 27,and a vessel 30 (often referred to as a cuvette) between the source andphotodetector. The filtrate sample being measured is placed in thecuvette 30. FIG. 1 illustrates the cuvette 30 as a discrete vessel, butit will be understood that it could also include a portion of tubing ora small reservoir or any other appropriate functional item, provided ithas the required transparency (minimal absorbency) in the color regionsmeasured by the colorimeter. For example, in the embodiments illustratedin FIGS. 7-13 the optics are positioned above the vessel 14 and theoptics pump 25 draws filtrate from the cup 10 and the filter 21 up intothe calorimeter 22.

As well understood with respect to protein dye reactions, the absorbanceof the filtrate follows Beer's Law, so that the measured color will beproportional to the concentration of dye in the filtrate sample. For thesame reason, the source 26 is selected to emit light in the frequencies(color) that the filtrate will absorb and the detector 27 is likewisesensitive to the relevant frequencies. As noted earlier, Orange 12 dyehas a characteristic absorption peak at about 482 nm.

In brief summary, Beer's Law states that the absorbance of a solutionvaries linearly with both the cell path length and the filtrateconcentration according to the formula A=e l c, where “e” represents themolar absorptivity (sometimes referred to as the extinctioncoefficient), “l” represents the cell path length and “c” represents theconcentration. The molar absorptivity varies with the wavelength oflight used in the measurement.

A processor 31 is in signal communication with the calorimeter 22through the line 32 which can represent a wire, a circuit board, or anyother appropriate means of transmitting the data from the calorimeter 22to the processor 31. The processor 31, which typically has thecapabilities of a personal computer, includes appropriate memoryschematically illustrated 33. Together, the processor 31 and the memory33 store the absorbance results from both reference and sample tests,compare the absorbance of the reference and sample tests, and calculatethe protein content of samples based on the comparisons. Because theprotein content is based upon weight, the processor is also linked tothe scale 19 through the line 38. 55.1 The use of processors and relatedelectronic circuits to control instruments based on selected measuredparameters (e.g. temperature and pressure) is generally well understoodin this and related arts. Exemplary (but not limiting) discussionsinclude Dorf, The Electrical Engineering Handbook, Second Ed. (1997) CRCPress LLC.

A display 34 is in communication with the processor through the line 35which again can be part of an integrated circuit as well as aconventional wire or similar electronic connection. The display can beused in any conventional manner with the processor 31, and in theinstrument according to the invention has the capacity to display itemssuch as the absorbance of a particular sample in the calorimeter 22 andthe protein content of a sample analyzed by the instrument. Althoughillustrated as a display, the instrument can include other forms ofoutput including a printer, or digital output to memory, or anotherdevice. The display is, however, most typical for bench top use. Theinstrument can, of course, concurrently support a plurality of outputformats.

FIG. 1 also illustrates some additional features that are included inexemplary embodiments of the instrument. A wash reservoir 35 is in fluidcommunication with the homogenizer 11 through the line 36 for providingthe homogenizer with a washing fluid, typically either de-ionized wateror a washing solution or sequential combinations of washing solutionsand de-ionized water. It will be understood, of course, that more thanone reservoir can be used for cleaning purposes. In some embodiments aheater 38 and filter 39 can be positioned between the wash reservoir 35and the homogenizer 11. In turn, the homogenizer 11 is connected to awaste schematically illustrated at 37 which can be a drain or containeror any other appropriate item. In the same manner, a wash reservoir 42can be in communication with the calorimeter 22, and in particular thecuvette (or equivalent) 30 for cleaning the cuvette 30 between samplemeasurements. A corresponding waste 43 is likewise in communication withthe cuvette 30 for completing a washing cycle. Depending upon thedesired design for fluid flow, a common wash reservoir can be includedin place of the separate reservoirs 35 and 42.

FIG. 1 also illustrates ports or openings 40 and 41 respectively thatare provided to permit either the protein sample or cup 10 to beinserted into the device or in order to facilitate adding water or dyeconcentrate to the reservoir 16.

In another embodiment, the automated protein analyzer is a combinationof (and with fluid communication between and among) the reservoir 16 forthe dye binding composition, the protein dye reaction vessel 14, thecalorimeter 22, and the pump 20 which is in fluid communication with thereservoir 16 and at least one of the calorimeter 22 and the reactionvessel 14 (and preferably both) for transferring dye compositions fromthe reservoir 16 to at least one of the calorimeter 22 or the reactionvessel 14. The processor 31 is in signal communication with thecalorimeter 22 for receiving the absorbance output from the calorimeter22 and the memory 33 is in signal communication with the processor 31for storing output from the calorimeter that includes (but is notlimited to) absorbance.

In this embodiment, the processor can compare the baseline absorbance ofa reference binding dye composition to the specific absorbance of a dyefiltrate solution following reaction with a protein to thereby calculateand determine the amount of protein in the protein sample based upon thedifference between the absorbance of the reference dye—i.e., directlyfrom the reservoir 16 and before the protein reaction—and the absorbanceof the dye filtrate remaining after a reaction with a protein sample.

FIGS. 2-4 illustrate a filter holder 45 for use as just described and inaccordance with the embodiments of the invention illustrated in FIGS.7-13. The cross-sectional view of FIG. 2 illustrates that the filterholder 45 includes a substantially planar filter medium 46 maintainedbetween an upper housing 47 and a lower housing 50. The housing portions47 and 50 together define a filtrate passage 51 axially through thefilter holder 45. The lower housing 50 defines a spout that depends fromthe filter medium 46. In use, the depending spout reached bottomportions of a sample cup 10 (FIGS. 5 and 6) when the filter medium 46 ispositioned above the cup 10. As will be further understood with respectto FIGS. 7-13, because the depending spout reaches lower portions of thecup 10, it helps encourage liquid, rather than protein solids or the dyeprecipitate from clogging the filter medium 46. In an exemplaryembodiment, the filter medium is a plastic scrim (for structuralsupport) combined with glass fibers.

Thus, in another embodiment the invention is a kit that includes thesample cup 10 and the filter holder 45. In exemplary embodiments, and asillustrated in FIG. 16, the kit (broadly designated at 78) includes aplurality of cups 10 and holders 45 (e.g., 50 of each) along with acontainer 80 of dye binding solution and a container 81 of washsolution. The amount of dye binding and wash solutions provided in thekit 78 is sufficient to carry out a number of tests equivalent to thenumber of cups 10 and filter holders 45.

FIGS. 5 and 6 illustrate a sample cup 10 (representing the same elementas in the schematic view of FIG. 1) used in accordance with theembodiments illustrated in FIGS. 7 and 8. Both the filter 45 holder andthe sample cup 10 can be formed of polymers making them easy tomanufacture, light weight, low cost, and minimally waste generating, allof which makes them suitable for use as consumable items. Beingconsumable, the need to clean them between uses can be eliminated andthe possibility that prior uses will contaminate the results of anygiven test can be eliminated.

FIGS. 7-14 illustrate one embodiment of a protein analyzer according tothe present invention. Most of the features will be described withrespect to FIGS. 7 and 8, and it will be understood that the same itemsappear in FIGS. 9-13 even if not specifically re-described.

FIGS. 7-13 specifically illustrate a series of stages (or steps) thattogether define one protein analysis cycle using this particularembodiment. FIG. 7 illustrates the first stage. In commercialembodiments the illustrated elements will typically be surrounded byhousing, but FIGS. 7-13 avoid including extraneous items for purposes ofclarity. Accordingly, FIG. 7 shows a platform 52 that supports aturntable 53 and a vertical translator 54. The vertical translator 54includes a horizontal arm 55 that carries the homogenizer 11 and thecalorimeter 22.

The homogenizer 11 includes a motor portion 56 and a blade shaft 57.

A motor 60 and related controls operate the vertical translator 54.Another motor and associated pulleys illustrated together at 61 drivesthe turntable 53.

The turntable 53 includes three stations: the wash station 62 shown asthe vertically oriented open cylinder, a cup holder 63 and the filterrest 64.

FIG. 7 illustrates the turntable 53 in the home position before anoperator places the cup 10 (and its sample) and the filter 45 in theirrespective holders.

FIG. 8 illustrates the same orientation as FIG. 7, but with the samplecup 10 in the sample cup holder 63 and the filter holder 45 in thefilter rest 64. In bench top operation, an operator will typicallyposition the cup 10 in the cup holder 63 and the filter holder 45 in therest 64. This is exemplary, however, rather than limiting of theinvention and these steps could be automated as well.

FIG. 9 illustrates the third stage of the operation in which theturntable has rotated to the position at which the binding dye is addedto the sample cup. The dye is added through a dye addition tube 65 thatin the illustrated embodiment is posited behind the calorimeter 22. Inthis position the sample cup 10 is under the dye addition tube 65. Inone embodiment, the turntable 53 can also include means (not visible inFIG. 9) for individually rotating the cup holder 63 on the turntable 53in order to rotate the cup 10 as the dye is being added. This additionalrotation helps mix the sample and dye in the cup 10.

FIG. 10 illustrates the fourth stage of the process which can bereferred to as the homogenization position. In this position, the cup 10is positioned under the homogenizer 11 and its blade shaft 57. Thefilter 45 is in turn positioned under the colorimeter 22. FIG. 10 alsoshows that in this position the vertical translator 54 has lowered theposition of the horizontal arm 55 to position the blade shaft 57 in thecup 10. At the same time, the optics tube 66 engages the top of thefilter holder 45 to temporarily fix the filter holder 45 to thecalorimeter. The sample is homogenized in this position.

FIG. 11 shows the fifth stage in the process in which the verticaltranslator 54 has raised the calorimeter 22 and the homogenizer 11 sothat the blade shaft 57 is above the cup 10 and the filter holder 45,still engaged to the optics tube 66, has likewise been raised above thefilter rest 64.

FIG. 12 shows the sixth stage in the process which represents thesampling position. The turntable 53 has rotated clockwise (with respectto FIGS. 11 and 12) to position the cup 10 with its sample underneaththe calorimeter 22 and to align the homogenizer 11 with the cleaningstation 62. When, as illustrated in FIG. 12, the vertical translator 54lowers the horizontal arm 55, the blade shaft 57 is likewise loweredinto the cleaning station 62 and the filter holder 45 is lowered intothe sample cup 10. In this position, the sample pump draws the sample upinto and through the filter holder 45. The filter media (e.g., 46 inFIGS. 2 and 4) allows only filtrate to reach the calorimeter 22. Thecalorimeter 22 then takes the absorbance reading in an otherwise wellunderstood manner. As set forth in the description of FIG. 1, in thisstep deionized water or another solution can be added to the cleaningstation 62 to clean the homogenizer 11, and in particular the bladeshaft 57. In this embodiment the pump (not shown) is positioned upstreamof the calorimeter 22 and in fluid communication with the supply ofde-ionized water or cleaning solution (e.g. the wash reservoir 42 inFIG. 1). This arrangement permits the pump to draw samples into thecalorimeter while handling only deionized water. In this embodiment, thepump can also run in the opposite direction to flush the sample from thecalorimeter 22.

FIG. 12 also illustrates the advantage of the filter holder 45. In thesampling position illustrated in FIG. 12, the filter holder 45 isinserted into the sample cup 10. Although not illustrated in FIG. 12,those familiar with the step of homogenizing a dye-binding solution witha solid protein (e.g., meat) recognize that the homogenization and dyereaction tend to generate a significant amount of foam along with thedye-protein precipitate and the remaining solids in the meat sample.

Because the filter holder 45 includes the spout 50 that complements thesize and shape of the cup 10 (FIGS. 15 and 16), the sample holder 45tends to avoid drawing excess solids to the filter. As a result, thefilter holder and cup arrangement encourages a freer flow of liquid tothe filter and a correspondingly better flow of filtrate from the filterinto the colorimeter. In one aspect, this arrangement of the cup 10 andfilter holder 45 eliminates the need for the pre-homogenizing dilutionstep with methanol and citric acid that is characteristic of certainconventional dye-binding techniques. In turn, eliminating the dilutionstep eliminates any potential error introduced with the dilution stepand also facilitates the automation of the process using the instrument

FIG. 13 illustrates the seventh and completion stage of the process ascarried out with this embodiment. The optics tube 66 ejects the filterholder 45 into the cup 10 and the optics are flushed with an appropriateliquid that can collect in the cup 10. Thus, at the end of this stage,and when the vertical translator 54 again raises the horizontal arm 55,the original sample, the used filter holder 45, and the rinse from thecalorimeter 22 are all in the cup 10. These items can be easily disposedof together. Removing the used sample cup 10, the used filter holder 45and the waste solutions returns the instrument to the first stageorientation and ready for the next sample as originally illustrated inFIG. 7.

In this regard, FIGS. 2-4 illustrate an exemplary embodiment in whichthe upper housing 47 of the filter holder 45 includes a male seal 70 andan ejector sheath 71. This combination cooperates with the optics tube66 to facilitate the engagement with, and the disengagement of, theoptics tube 66 and the filter holder 45.

FIG. 15 is a perspective view of the instrument broadly designated at 68in the context of its housing broadly designated at 73. A keyboard oranalogous control panel 74 can be used to provide relevant instructionsto the device and operates in combination with a display 75. As notedearlier, the nature of individual control panels, processors,controllers, and displays is generally well understood in this art andwill not be described in detail.

In the embodiment illustrated in FIG. 15, the housing 73 includes agenerally cylindrical portion 76 which in this embodiment represents theposition of the turntable 53 and the associated elements of theinstrument that are illustrated in FIGS. 7 through 13. The cylindricalhousing portion 76 includes a door 77 that can be opened and closed toposition new samples on the turntable 53 or remove analyzed samples fromthe turntable 53.

In another aspect, the invention is a dye binding method for proteinanalysis. In this aspect, the invention comprises preparing an initialreference dye solution of unknown concentration from an initialreference dye concentrate. Reference dye concentrates of knownconcentration are available in the art, but as described further herein,the method and instrument of the invention can eliminate some of themeasuring steps that conventional methods require. Typically, areference dye solution can be prepared by diluting a reference dyeconcentrate with water, and potentially items such as a weak acid (e.g.,acetic) to adjust pH or an alcohol (e.g., ethanol) to reduce foaming.

The method next includes the step of creating an electronic (e.g.,digital or analog) signal based upon the absorbance of the initialreference dye solution. The absorbance is used herein in itsconventional sense to refer to a Beer's Law analysis as discussedearlier with respect to the instrument.

In the following step, the method includes creating an electronic signalbased upon the absorbance of a dye filtrate solution prepared from theinitial reference dye solution and an initial protein sample. Theabsorbance signals from the reference dye solution and the dyed filtratesolution are respectively sent to a processor that compares therespective absorbances and calculates the protein content of the proteinsample based upon the differences between the absorbances.

The method next includes the step of creating an electronic signal basedupon the absorbance of a successive dye filtrate solution prepared fromthe reference dye solution and a successive protein sample.

The absorbance signal from the successive sample dye filtrate solutionis also sent to the processor to calculate the protein content of thesuccessive sample based upon the difference between the absorbance ofthe initial reference dye solution and the absorbance of the successivedye filtrate solution.

The method can further comprise the step of weighing a protein sampleand mixing the sample with the initial dye reference solution prior tocreating the electronic signal based upon the dye filtrate solution. Inturn, the step of weighing the protein sample can comprise adding thesample to a tared sample cup and weighing the cup and the sample.

The method can further comprise the step of homogenizing the sampleafter the step of weighing the sample. This in turn particularlydistinguishes the method and instrument of the invention from priortechniques in which the homogenization of the sample is typicallycarried out prior to the step of weighing the material. The step ofhomogenizing the sample can be selected of the group consisting ofgrinding, pulverizing, blending, milling, and combinations thereof.

After the sample has been homogenized, the method can comprise mixingthe reference dye solution with the homogenized sample by physicallyagitating the dye solution and the sample. The method includes filteringthe mixture of the initial reference dye solution and the initialprotein sample prior to the step of creating the electronic signal basedupon the absorbance of the filtrate. In the same manner, the methodincludes the steps of filtering a mixture of a successive reference dyesolution and a successive protein sample prior to the step of creatingthe electronic signal based upon the absorbance of the filtrate.

Although the term “filtrate” nominally refers to a solution from whichsolids have been filtered, in the context of the present invention itcan be used to describe any post-reaction dye solution from which solidshave been separated. For example, centrifuging the solids from theprotein-dye mixture will produce an appropriate filtrate.

As a particular advantage, the method includes repeating the protein-dyeanalysis to produce successive dye filtrate solutions until the initialreference dye solution is exhausted. A successive reference dye solutionof unknown concentration is then prepared from a reference dyeconcentrate. The remaining analysis steps are then repeated forsuccessive dye filtrate solutions formed from successive reactionsbetween the successive reference dye solution and successive proteinsamples.

Once the initial reference by solution is exhausted, the successivereference dye solution can be prepared from the initial reference dyeconcentrate or from a different dye concentrate, because the method andrelated instrument provide the opportunity to recalibrate based on everyreference dye solution.

In another aspect, the invention comprises a method of calibrating acalorimeter for protein analysis and analyzing a plurality of proteinsamples. In this aspect the method comprises preparing an initialreference dye solution of unknown concentration from an initialreference dye concentrate and an approximate amount of liquid. As in theother embodiments, the liquid is primarily de-ionized water but can alsoinclude items such as acetic acid or ethanol.

The initial reference dye solution—without reacting with anything—isforwarded to a calorimeter where the absorbency of the initial referencedye solution is measured in a Beer's Law context. Thereafter, an initialdye filtrate solution that has been prepared from the reaction of theinitial reference dye solution and a protein sample is forwarded to thecalorimeter and the calorimeter measures the absorbency of the initialdye filtrate solution.

The absorbance results from the initial dye filtrate solution and theinitial reference dye solution are sent to a processor that compares therespective absorbances and calculates the protein content of the samplebased upon the difference between the absorbances.

Then, a successive dye filtrate solution—from a successive dye-proteinreaction—is sent to the calorimeter and the absorbency of the successivedye filtrate solution is measured. The absorbance results from thesuccessive dye filtrate solution are sent to the processor to calculatethe protein content of the successive sample based upon the differencebetween the absorbance of the initial reference dye solution and theabsorbance of the successive dye filtrate solution.

In this method, the step of forwarding the sample dye solution canfurther comprise the steps of mixing a protein sample with a portion ofthe initial reference dye solution, then filtering the protein-dyeprecipitate generated when the protein sample reacts with the initialreference dye solution, and then forwarding the filtrate to thecalorimeter.

As in the other embodiments, the protein sample is typically homogenizedbefore being mixed with the initial reference dye solution.

The method can further comprise repeating the step of forwardingsuccessive dye filtrate solutions to the calorimeter until the initialreference dye solution prepared from the reference dye concentrate isexhausted. Then, a successive reference dye solution can be prepared bymixing a reference dye concentrate with a successive approximate amountof liquid to produce a successive working dye solution of unknownconcentration.

The method can further comprise repeating the steps of forwarding thereference dye solution, forwarding the dye filtrate solution, sendingthe absorbance results, forwarding the successive dye filtratesolutions, and sending the successive absorption results, all followingthe step of mixing the successive portion of liquid with reference dyeconcentrate.

As in the other embodiments, the successive reference dye solution canbe prepared from the initial reference dye concentrate or from adifferent reference dye concentrate.

FIG. 14 illustrates the manner in which the protein content of generalcategories of samples can be normalized so that the instrument providesconsistent results as the reference dye solution is used and thenreplenished. FIG. 14 also relates to Tables 1-4.

The purpose of normalization is to standardize the instrument so thatdifferent initial dye concentrations produce consistent protein contentresults. As noted previously, the dye binding technique dependsfundamentally upon the difference in absorbance (color density) betweenthe dye before it reacts with protein and after it reacts with protein.If the starting concentration (color) of the dye solution is increased(or decreased), then the color intensity after reaction will becorrespondingly greater (or less) for all protein samples tested withthat dye solution.

Stated in yet another fashion, a more concentrated starting dye solutionwill produce a more concentrated solution even after reaction with acertain amount of protein. In the same manner, a less concentratedstarting dye solution will produce a less concentrated solution afterreaction with a certain amount of protein. Thus, an identical proteinsample will give different calorimeter results based on differentstarting dye concentrations. Accordingly, the normalization stepcompensates for the difference in the initial dye solutions and producesa consistent output from the instrument.

Tables 1-4 illustrate one method for normalizing the results as betweentwo different initial dye solutions.

Table 1 presents data from a first dye solution arbitrarily designatedas “A.” The “A” dye solution is placed in the calorimeter to obtain itsabsorbance reading (28.56 in this example). A blank sample of de-ionizedwater is then immediately placed in the same calorimeter to obtain itstransmission (443.60). The absorbance (1.191) of the “A” initial dyesolution is then calculated according to the formulaAbsorbance=log(dye transmission/blank transmission).

Table 2 gives the results for four (4) samples of turkey paste using the“A” initial dye solution. In order to create a baseline for theanalysis, the turkey paste was first tested using the Kjeldahl method,which indicated a protein content of 31.23 percent by weight.

Four respective samples of this turkey paste were then tested using theinstrument and method of the invention (“Reading”). Each sample readingwas followed immediately by a reading of deionized water (“Blank”). Theabsorbance was calculated on this basis. The results were then plottedusing a least squares analysis to form the straight line illustrated inFIG. 14. The theoretical protein percent was then taken from the leastsquares line (“Theoretical Protein (%)”) and the comparisons to theKjeldahl results (“Error”) were calculated for each sample.

Table 3 represents a second initial dye solution designated as “B” whichwas purposefully made to a different concentration than the “A” initialdye solution. Its transmission, the transmission of a deionized waterblank, and the absorbance of the “B” solution were appropriatelycalculated.

The difference between the absorbance of the “A” initial dye solution(1.191) and the absorbance of the “B” initial dye solution (0.923)represents the extent to which results using the “B” initial dyesolution must be normalized to give results consistent with the “A”initial dye solution.

Table 4 shows these results for three more samples of the same turkeypaste. The raw results using the “B” dye solution are plotted as thesmall squares in the lower portion of FIG. 14. When the differencebetween the initial absorbances of the “A” and “B” solutions are addedto the “B” data points, the “B” data points fall as the triangles inFIG. 14. Because the triangles now fall along the least squares linecreated from the “A” solution, the results using the “B” solution can becompared directly to the results of the “A” solution.

This normalization step can be carried out in the same manner for thirdand succeeding concentrations of the initial dye solution thus providingthe instrument with the capability to provide consistent protein contentresults independent of normal variations in the concentration of initialdye solutions.

Turkey Paste Normalization Trial

TABLE 1 Tank Reading Blank A_(TANK) A 28.56 443.60 1.191

TABLE 2 Theoreti- Weight wt cal Protein Error Sample (g) protein ReadingBlank Abs (%) (%) 1 0.2096 0.0655 83.19 445.2 0.7285 31.11 0.12 2 0.24580.0768 100 444.9 0.6483 31.39 0.16 3 0.2992 0.0934 128.2 444.6 0.540131.17 0.06 4 0.3491 0.1090 163.4 444.2 0.4343 31.23 0.00

TABLE 3 Tank Reading Blank A_(TANK) B 52.78 442.30 0.923

TABLE 4 Normal- NORMAL- ized IZED A Theo (%) 1 0.2521 0.0787 190.5 442.30.3658 0.634 31.46 2 0.2002 0.0625 150 442.2 0.4695 0.738 31.90 3 0.3090.0965 245.3 441.9 0.2556 0.524 30.98

In the drawings and specification there has been set forth a preferredembodiment of the invention, and although specific terms have beenemployed, they are used in a generic and descriptive sense only and notfor purposes of limitation, the scope of the invention being defined inthe claims.

The invention claimed is:
 1. An automated protein analyzer comprising: areaction vessel for protein samples; a homogenizer above said reactionvessel for reducing protein samples in said reaction vessel to smallparticles; a reservoir for binding dye composition; a metering pumpconnected to said reservoir for distributing discrete predeterminedamounts of a binding dye composition from said reservoir to saidreaction vessel; a colorimeter on a platform that supports saidhomogenizer and said colorimeter above said reaction vessel formeasuring the absorbance of the binding dye composition from saidreaction vessel; and a filter above said reaction vessel and-betweensaid colorimeter and said reaction vessel and connected with saidcolorimeter for separating solids from filtrate after a dye bindingreaction has taken place in said reaction vessel.
 2. A protein analyzeraccording to claim 1 further comprising: a processor in signalcommunication with said colorimeter; and memory in signal communicationwith said processor for storing the absorbance results from referenceand sample tests, for comparing the absorbance of reference and sampletests, and for calculating the protein content of a sample based on thecomparisons.
 3. A protein analyzer according to claim 1 wherein saidhomogenizer is selected from the group consisting of blenders, mills,and combinations thereof.
 4. A protein analyzer according to claim 1further comprising: a valve between said reservoir and said reactionvessel; and a fluid line between said valve and said colorimeter; sothat binding dye solution from said reservoir can be directed to saidreaction vessel or to said colorimeter based upon the position of saidvalve.
 5. A protein analyzer according to claim 2 further comprising adisplay in signal communication with said processor for displaying itemsselected from the group consisting of absorbance and protein content. 6.A protein analyzer according to claim 1 further comprising a pump fortransferring filtrate from said reaction vessel to said colorimeter. 7.A protein analyzer according to claim 6 wherein said pump is upstream ofsaid colorimeter with respect to said reaction vessel.
 8. A proteinanalyzer according to claim 1 further comprising a wash reservoir incommunication with said homogenizer for providing said homogenizer witha washing fluid.
 9. A protein analyzer according to claim 8 furthercomprising a drain from said homogenizer for carrying waste washingfluid from said homogenizer.
 10. An automated protein analyzer accordingto claim 2 wherein: said processor compares the baseline absorbance of areference binding dye composition to the specific absorbance of abinding dye composition following reaction with a protein and calculatesthe amount of protein in a protein sample based upon the differencebetween the absorbance of the reference dye and the absorbance of thereference dye after it has reacted with a protein sample.
 11. A proteinanalyzer according to claim 10 further comprising a display in signalcommunication with said processor.